Influenza virus averages 30-50 million infections annually in the United States alone. Epidemiologic studies of influenza epidemics estimate the incidence of infection to be 25% in the general population and higher in school age children. Researchers have estimated that up to half the infected persons would see a physician because of the illness. In 1986, the Center for Disease Control (CDC) estimated that influenza epidemics have been associated with 10,000 or more excess deaths in 18 of the preceding 28 years. CDC studies indicate influenza as the fifth leading cause of death in the United States. Antigenic variations in the surface glycoproteins of influenza A and B and reassortment of viral proteins in influenza A account for their continued epidemics.
Influenza viruses possess surface glycoproteins that have NA activity. These glycoproteins are members of a family of neuraminidases that are found in viruses, bacteria, mycoplasmas, and animal tissues. They hydrolyze substrates that contain alpha-ketosidically linked N-acetylneuraminic acid (Neu5Ac; referred to previously as "NANA"). In viruses, NA typically constitutes 5-10% of the viral protein and exists as a mushroom-shaped spike on the envelope. Viral NA is composed of a hydrophilic area which includes the catalytic site of the enzyme and a hydrophobic area that is inserted into the viral envelope anchoring the enzyme to the virus.
Various assays for NA activity are described in the literature. Santer, U. V., et al., Biochimica et Biophysica Acta 523:435-442 (1978), describes a colorimetric assay for NA using 2-(3-methoxyphenyl)-N-acetyl-alpha-D neuraminic acid as a substrate and 4-aminoantipyrine in the presence of an oxidizing agent to measure the enzymatically released methoxyphenol. Myers, R. W., et al., Analytical Biochemistry 101:166-174 (1980), describes the use of the 4-methylumbelliferyl-alpha-ketoside of Neu5Ac in a fluorometric assay for NA. This fluorogenic derivative of Neu5Ac was also used in studies of the NA activity of influenza viruses by Yolken, R. H., et al., J. Infectious Diseases 142:5116-523 (1980); Clinical Chemistry 27:1490-1498 (1981); and Reviews of Infectious Diseases 4:35-68 (1982); and by Kiyotani et al., Hiroshima J. Medical Sciences 33:287-292 (1984); Zbl Bakt Hyg A260-273-285 (1985); Microbiol. Immun. 31:1131-1135 (1987). Despite the availability of these prior NA assays, however, physicians currently still diagnose influenza solely on the basis of symptomology. This is in part due to the fact that these prior assays were complicated and/or required equipment not typically found in a clinical setting. Another shortcoming of these prior assays is that they were unable to discriminate between influenza type. That ability is particularly important to enable physicians to prescribe the appropriate chemotherapy and/or supportive therapy to combat the infection.
Prior workers have investigated the relationship between the chemical structure of Neu5Ac and its biological function as a substrate for non-influenza NA. Gross, H. J. et al., Biochemistry 27:4279 (1988), examined benzyl-alpha-glycosides of N-acetyl-4-epi-D-neuraminic acid as a substrate for three different bacterial NAs (C. perfringens, A. ureafaciens, and V. cholera) and found significant differences in reactivity. After 22 hours, the C. perfringens NA cleaved 100% of the substrate while the A. ureafaciens and V. cholera NAs cleaved only 50% and 11% of the substrate, respectively. Kim et al., J. Am. Chem. Soc. 110:6481-6486 (1988) described the structural characteristics of substrates accepted by Neu5Ac aldolase, its use in the synthesis of Neu5Ac, and its chemical conversion to the 2-deoxy derivatives, and additionally reported that work was in progress to determine the biological activity of the 2-deoxy derivatives. Brossmer et al., Helv. Chim. Acta 69:2127 (1986); Glycoconjugates 4:145 (1987) reported that the methyl-alpha-glycoside of 4-deoxy Neu5Ac was a good substrate for fowl plague viral Neu5Ac, but not for the three bacterial NAs mentioned above. Additionally, Schauer, R., et al., Eur. J. Biochem. 106:531 (1980), reported that 4-methoxy Neu5Ac was an excellent substrate for fowl plague viral NA but not for V. cholera NA. The 4-methylumbelliferyl derivative of 4-deoxy Neu5Ac is also described in the literature (Helv. Chim. Acta. 69:1927 (1986)). Zbiral et al., Monatsheft fur Chemie 119:127-141 (1988) described the synthesis of 7- and 8-deoxy Neu5Ac and 4,7-dideoxy Neu5Ac. Zbiral et al., Liebigs Ann. Chem. 119:127-141 (1989) described the synthesis of the 4-methylumbelliferyl-2-.alpha. glycosides of 7-epi, 8-epi, 7,8-bis-epi, 8-deoxy, 9-deoxy and 4,7-dideoxy Neu5Ac and investigated the behavior of those compounds as inhibitors of the sialidase from V. cholera.
Applicant is unaware of any prior reports on the reactivity of 4-modified Neu5Acs with human influenza NA.